Optical sensing devices with spr sensors based on differential phase interrogation and measuring method using the same

ABSTRACT

Disclosed is an optical sensing device, which comprises a light source emitting a light; a beam splitter; an SPR sensor unit comprising a sensing surface; and a detecting mechanism; and a converting unit converting the first beam and the second beam from the optical device into a two-dimensional interference fringe pattern. From the above-mentioned configuration, an extra phase shift of a detection beam in SPR phase measurement is obtained. The differential measurement approach has shown to achieve a sensitivity figure significantly better than the best result that can be obtained from the prior art in the field of the measurement based on an SPR sensor.

CROSS-REFERENCE TO RELATED APPLICATIONS

This patent application is a Divisional of U.S. patent application Ser.No. 11/178,077 filed on Jul. 8, 2005, to which priority is claimed, andthe entire contents of which are hereby incorporated by reference.

TECHNICAL FIELD OF THE INVENTION

This invention relates to surface plasmon resonance (SPR) sensors, andparticularly to optical surface plasmon resonance sensors based ondifferential phase interrogation and phase enhancement using aFabry-Perot cavity for detection of a molecular species.

BACKGROUND OF THE INVENTION

Various types of optical sensors utilizing SPR measurement have beenreported since the first gas detection and biosensing sensor based onSPR was developed by Nylander et al., “Surface plasmon Resonance for gasdetection and biosensing” by Lieberg, Nylander and Lundstrom in Sensorsand Actuators, Vol. 4, page 299. Traditional sensing technique forextracting information from SPR is primarily concerned with analyzingangular and wavelength properties of a reflected light beam within theresonant reflectance dip.

Nelson et al. presented the first SPR sensing based on optical phasedetection (Sensors and Actuators B, 35-36, 187-191, 1996). The phasequantity has a steep slope response and therefore offers the potentialfor at least 3 times greater resolution than sensors based onconventional angle and wavelength modulation.

However, Nelson's phase detection system is very sensitive to mechanicalvibrations and phase errors incurred in the system may becomeproblematic. System stability issues of Nelson's system limit itsapplications. Phase detection improvements under “noisy” environmenthave been widely studied.

Recent research's attention to the SPR sensing has shifted to measuringthe SPR phase shift, because the resonant phase behavior exhibits asteep jump, which leads to the potential in achieving extremely highsensitivity.

The most recent work was presented by Chien-Ming Wu et al., who reporteda heterodyne interferometric system for the investigation of phasevariation during surface plasmon resonance (Sensors and Actuators B 92,133-136, 2003). The system used a combined SPR with total internalreflection (TIR) so that an extra phase shift caused by TIR may be addedto a detected signal. In addition, a reference optical path is employedin the system to suppress unwanted noise. But the drawback is that thesensor response is measured by monitoring the phase shift versusincident angle, which requires highly stable mechanical parts. Also, thereference optical path is not totally identical to the signal path.Therefore, common-mode noise may still exist in the differentialmeasurement.

Sheridan analyzed the phase behavior of an SPR waveguide sensor (Sensorsand Actuators B 97, 114-121, 2004). The system employed a Mach-Zehnderinterferometer (MZI) configuration with two sensing branches. Since onlyone branch was exposed to index variation, unwanted refractive indexchanges common to two branches such as temperature variation are said tobe suppressed. This device was found to provide a higher sensitivitythan the conventional single-waveguide configuration.

FIG. 6 shows a conventional sensing device with an SPR sensor of theKretschmann configuration, of which the detailed description anddiscussion can be found in the articles by H. P. Ho, et al., “Real-timeoptical biosensor based on differential phase measurement of surfaceplasmon resonance” (BIOSENSORS & BIOELECTRONICS, 20 2177-2180, 2005),and by S. Y. Wu, et-al., “Highly sensitive differential phase-sensitivesurface plasmon resonance biosensor based on the Mach-Zehnderconfiguration” (OPTICS LETTERS 29, 2378-2380, 2004). Their entiredisclosure is incorporated herein by reference.

As shown in FIG. 6, a light beam from an He-Ne laser source 1′ isdivided into two beams Is and Ir by a 50:50 beam splitter 2′. In asignal path, the beam Is enters a silver sensing surface 41′ of asensing head 4′, while the beam Ir is reflected by a mirror surface 3′in a reference path. The two beams are recombined at a beam splitter 5′and form interference pattern. After passing through a Wollaston prism6′, SPR images are captured by a detection device 7′, and then are inputto a data processor 8′ for analysis.

Although the differential SPR phase approach as shown in FIG. 6 hasimproved measurement accuracy significantly, similar to other SPR phasetechniques, the problem existing in the SPR phase approach is that thedistribution of SPR phases over the sensing surface is not recorded. Theinformation obtained from the mapping of surface reactions which can bedirectly inferred from the SPR phase distribution is necessary andimportant for many bio-reaction monitoring applications. For example,users may want to perform biosensing of a range of analytes from asingle sample simultaneously. The capability of the SPR phase imaging isparticularly important. In addition, given that the phase imagingimposes more stringent requirements on an optical instrument due to arelatively low signal to noise ratio and a low frame rate (i.e. low datacollection speed) achievable by a common imaging device such as CCDs, itis necessary to further develop the current differential SPR phasetechnique, in order to provide a better signal to noise ratio for theimaging measurement, while enabling the phase to be stepped as requiredat a speed appropriate to the imaging device.

SUMMARY OF THE INVENTION

In general, the present disclosure describes an optical sensing devicehaving a sensing surface, which can enhance the phase shift of anincident beam caused by the SPR effect, for example, by permitting theincident beam to pass through the sensing surface at least twice.

In exemplary embodiments, the present disclosure describes methods ofdetecting biological, biochemical, or chemical characteristics of aspecies.

To achieve the objects aforementioned, according to one aspect of thepresent invention, there is provided an optical sensing devicecomprising:

a light source emitting a light;

a beam splitter configured to divide the light into a first beam and asecond beam, wherein the beam splitter allows the first beam to passtherethrough while reflecting the second beam;

an SPR sensor unit comprising a sensing surface, wherein the first beamfrom the splitter is reflected by the sensing surface at least twice andbe sent back to the splitter; and

a detecting mechanism comprising:

a reflecting unit reflecting the second beam from the beam splitter andthe first beam coming from the sensor unit through the beam splitter,and

a converting unit converting the first beam and the second beam from thereflecting unit into a two-dimensional interference fringe pattern.

According to an embodiment of the invention, the optical sensing deviceof the invention further comprises a data processor for analyzinginterference patterns detected by the detecting mechanism.

According to embodiments of the invention, the sensor unit comprises a60° equilateral SF18 glass prism which is coated with a metal layer. Asensitive material may be placed on the metal layer directly or throughan intermediate material. The sensitive material can be an antibody thatbinds complementarily a corresponding antigen. A flow channel made fromchemically inert Teflon may be attached to the surface of the sensitivematerial. It should be noted that any chemically and biological inertmaterial in addition to Teflon can also be used for the flow cell.

According to another aspect of the invention, there is provided a methodof detecting biological, biochemical, or chemical characteristics of aspecies, comprising: providing a device comprising an SPR sensor havinga sensing surface;

allowing a fluid including the species through a channel attached to thesensing surface;

subjecting a light to passing through the SPR sensor so that the lightis reflected at least twice by the sensing surface;

obtaining a two-dimensional interference fringe pattern of the lightfrom the SPR sensor;

measuring a differential phase between a p-polarization and ans-polarization of the light through analyzing the two-dimensionalinterference fringe pattern; and

determining the biological, biochemical, or chemical characteristics ofthe species in accordance with the differential phase.

According to one embodiment of the method, the step of measuring thedifferential phase comprises:

averaging an interference fringe pattern of the light over a region toproduce a profile of an intensity variation for each of the p- ands-polarization interference fringe patterns;

extracting an intensity distribution of each of the p- ands-polarization interference fringe patterns from the profile to obtain acurve of the intensity distribution of each of the p- and s-polarizationinterference fringe patterns;

differentiating the curve to find a peak of each of the curves; and

obtaining the differential phase through a location of the peak of eachof the curves.

According to still another aspect of the invention, there is provided anoptical sensing device, comprising:

a light source emitting a light;

a beam splitter configured to divide the light into a first beam and asecond beam, wherein the beam splitter allows the first beam to passtherethrough while reflecting the second beam;

an SPR sensor unit magnifying a phase change of the first beam from thebeam splitter; and

a detecting mechanism comprising:

a reflecting unit reflecting the second beam from the beam splitter andthe first beam coming from the SPR sensor unit through the beamsplitter, and

a converting unit converting the first beam and the second beam from thereflecting unit into a two-dimensional interference fringe pattern.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram of an optical sensing device with an SPRsensor of this invention;

FIG. 2 a is an SPR sensor interference fringe pattern image obtained inan air detection;

FIG. 2 b is a graph showing corresponding intensity distributions of thes- and p-polarized beam;

FIG. 3 is a block diagram for illustrating the fringe analysis methodaccording to the invention;

FIG. 4 shows the SPR fringe patterns of salt-water mixtures with varioussalt concentrations;

FIG. 5 is a graph showing the quantitative differential phase withrespect to refractive index, which is obtained from the sensing deviceof the invention;

FIG. 6 is a diagram showing the schematic layout of a conventionalsensing device based on a Mach-Zehnder interferometer (MZI);

FIG. 7 shows the SPR fringe patterns of salt-water mixtures of varioussalt concentration obtained by conventional sensing device as shown inFIG. 6;

FIG. 8 is a graph showing the quantitative differential phase obtainedfrom a conventional sensing device as shown in FIG. 6 with respect torefractive index, and

FIG. 9 is a graph showing the phase response enhancement resulted fromthe invention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention and various advantages thereof will be describedwith reference to exemplary embodiments in conjunction with thedrawings.

One aspect of the invention provides an optical sensing device,comprising: a light source emitting a light; a beam splitter configuredto divide the light into a first beam and a second beam, wherein thebeam splitter allows the first beam to pass therethrough whilereflecting the second beam; an SPR sensor unit magnifying a phase changeof the first beam from the beam splitter; and a detecting mechanismcomprising a reflecting unit reflecting the second beam from the beamsplitter and the first beam coming from the SPR sensor unit through thebeam splitter, and a converting unit converting the first beam and thesecond beam from the reflecting unit into a two-dimensional interferencefringe pattern.

An optical sensing device according to one embodiment of the presentinvention is shown in FIG. 1. The device comprises an excitation lightsource 1 such as a random polarization He—Ne laser source, which emits abeam I0. An optical device 2 is provided in a path of the beam I0 fromthe light source 1, and disposed at an angle of 45° with respect to thepath. An SPR sensor unit 45 is provided downstream in the path toreceive the beam I0. A beam splitter 3 is provided between the opticaldevice 2 and the unit 45.

The beam splitter 3 separates the beam IO passing through the opticaldevice 2 into two beams Is and Ir, the beam Is passing through thesplitter 3, while the beam Ir being reflected back to the optical device2.

The SPR sensor unit 45 comprises a sensing surface 40 which is disposedin a manner that the beam from the splitter 3 can be reflected by itselfat least twice. According to an embodiment of the invention, as shown inFIG. 1, the SPR sensor unit 45 comprises an SPR sensor 4 and areflecting mirror 5.

In one preferred embodiment, the SPR sensor is a 60° equilateral SF18glass prism 41, which is disposed as shown in FIG. 1. In thisembodiment, the mirror 5 is disposed to reflect the beam reflected bythe sensing surface 40 back to it. Then the beam reflected by the mirroris reflected by the sensing surface 40 back to the splitter 3. In such amanner, the beam passing through the splitter 3 is reflected twice bythe sensing surface and sent back to the splitter.

In this embodiment, the 60° equilateral SF18 glass prism 41 is coatedwith a metal layer 42. A sensitive material 43 is further placed on thesurface of the metal layer 42. The metal layer 42 can be formed by firstsputtering a silver layer of about 48 nm on the glass prism 41 toprovide a sensing surface 40 and then sputtering a gold layer of about 3nm (not shown) on the silver layer to be a protective layer for thesensing surface of the silver layer. The sensitive material 43 can be anantibody that binds complementarily a corresponding antigen. Inaddition, a channel 44 made from e.g. chemically inert Teflon isattached to the surface of the sensitive material 43.

The light beam I0 from the light source 1 first passes through theoptical device 2 without attenuation and then is divided into two beamsby the beam splitter 3: one is the penetrated beam Is to be used as adetecting light and the other beam is the reflected beam Ir to be usedas a reference light. Then, the beam Is enters the SPR sensor 4. At aresonance incident angle, the beam Is enters the sensing surface 40first time to form a beam Is1 which experiences a first phase shiftcaused by surface plasmon resonance effect. Then the beam Is1 isreflected by the sensing surface 40. By providing the mirror 5 at theend of the optical cavity, the beam Is1 is reflected and enters thesensing surface 40 again (second time) at the same incident angle. Anextra phase shift is thereby added to the beam Is1. Then the beam Is1passes through the beam splitter 3 again (as beam Is2). Finally, thepenetrated beam Is2 recombines with the reference beam Ir to form arequired interference signal.

It should be noted that, although in the above embodiment, the phasevariance of the beam Is1 caused by SPR effect is enhanced by using themirror 5 to bounce back the beam Is1 reflected from the sensing surface,other manners for enhancing the phase variance are readily conceivableto those skilled in the art.

A Wollaston prism 6 for separating a polarized optical interferencesignal beam into a p-polarization component and an s-polarizationcomponent is placed in front of a digital two-dimensional array detector7 such as a CCD camera to receive the combined beam from the opticaldevice 2. Since only a p-polarization light is affected by the SPReffect, and an s-polarization light is served as a reference signal, theabove optical system can be considered as two independentinterferometers operating in parallel. The advantage of thisconfiguration is that any unwanted common mode phase drift caused bymechanical, temperature or other kind of variations can be eliminatedthrough measurement of the differential phase between p- ands-polarization signals. Finally, two interference fringes patternrelated to both the polarizations are captured by the digital CCDcamera.

The captured patterns are input into a data processor 8 to be analyzedby a fringes analysis process, for which a program written by e.g. MathLab can be used. An embodiment of the fringes analysis process will bedescribed hereinafter with reference to FIG. 3.

Referring to FIG. 3 now, in this process, the interference patterns areread from a data source (S101) and the data format thereof is identified(S102). If the interference patterns are in a video format, then theyare separated into frames (S103). The frames are triggered to be storedin variable (S104) and then are averaged (S105). On the other hand, ifthe interference patterns are a set of single images, they will also beaveraged (S105). The averaging process involves first reading intensityvalues of pixels in a pre-defined rectangular region within an image.The rectangular region is then separated into one-pixel wide verticaltraces each containing a different intensity value. These traces areseparately averaged by finding a mean horizontal pixel value of each ofall the vertical traces. Then a profile of the intensity variation of aninterference signal is produced by the mean horizontal pixel values forthe image. Effectively a two-dimensional interference pattern hasfinally been reduced to a one-dimensional array representing theaveraging of the pattern. As shown in FIG. 2( a), for example, the tworegions enclosed by the boxes have been averaged to produce two tracesof interference signal. These traces will later be used for calculatinga differential phase between the two orthogonal polarizations. Theaveraging operation is used to increase the stability of the intensitydistribution curve extracted from the image. This provides a moreaccurate phase measurement. Then, the intensity distribution for each ofthe p- and s-polarization interference fringe patterns is extracted foreach frame of images.

FIG. 2 b is an intensity distributions plot for air sensing. A FastFourier Transform (FFT) followed by a low pass filtering is conductedfor smoothing the curve of the intensity distribution (S106), which willresults in a more accurate differential phase information.

The smoothed intensity distribution curve is differentiated (S107).Then, a peak in the distribution curve as represented in a pixellocation is found by differentiating the curve (S108). In addition, astandard deviation of differential phase measurement between two framescan also be used to indicate a system measurement error. Then, whetheror not the location of the peak is an integer pixel value is determined(S109).

If the location of the peak is an integer pixel value, the differentialphase between the p- and s-polarization intensity distribution curves isobtained by comparing the location of the peak of the two sinusoidalwaveforms represented in the pixel location (S111). If it is not aninteger pixel value, the peak will be located as a fraction of pixel(sub-pixel) by means of linear extrapolation using the intensity valuesof two adjacent pixels (110). Thus, the differential phase is obtainedaccordingly (S111).

Then, the differential phase value is stored according to the dataformat of the interference patterns (S112). If the interference patternsare in single image format, the differential phase value will be stored(S113). If the interference patterns are in video format, then thecalculated differential phase will be stored in the kth position in adata array (S114). Then, the interference pattern of the next frame willgo through step (S104) to step (S114). Finally the data array ofdifferential phase values is obtained.

EXAMPLE 1

To demonstrate the performance of the present invention, salt-watersensing experiments were carried out using the device as shown inFIG. 1. A group of salt-water mixture samples were used. Theconcentrations of the samples were 0 wt %, 0.5 wt %, 1 wt %, 2 wt %, 3wt %, 5 wt %, and 7 wt % respectively with the corresponding refractiveindex ranging from 1.3333 to 1.3453. A series of interference fringepattern images obtained from the samples were recorded and shown in FIG.4.

After processed by the fringe analysis method as mentioned above,quantitative differential phase values were obtained and plotted in FIG.5. The phase response was found to be 6.1×10=5 RI/degree with anuncertainty of 1.7×10−2 rad. The calculated system sensitivity was1.2×10−4 RIU.

In order to show the phase measurement enhancement by the presentinvention, a conventional SPR phase detection set-up based on theMach-Zehnder interferometer as shown in FIG. 6 was used to repeat themeasurement of the salt-water mixture samples. As explained previously,in this conventional configuration, the light beam only entered thesensing surface once and no extra phase shift was present. Measurementwas conducted for salt-water mixture samples with concentrations being 0wt %, 0.5 wt %, 1 wt %, 2 wt %, 3 wt %, 5 wt %, and 7 wt % respectivelyby using the same analysis method. The interference fringes patternimages were shown in FIGS. 7 a-7 h. Corresponding differential phasevalues were plotted against refractive index values in FIG. 8 and werefurther compared with previous results in FIG. 9.

It can be seen from FIG. 9 that the rate of change of the differentialphase shift for the phase enhanced SPR sensor according to the presentinvention is much steeper than that for the conventional SPR device.Particularly, the experimental results obtained from the salt-watermixture samples indicate that the phase response for the conventionaldevice is about 1.5×10−4 RI/degree, compared with that for the phaseenhanced SPR sensor of the invention, namely, 6.1. In other words, thepresent invention has enhanced the sensitivity by 2.8 times. This is asignificant improvement over previously obtained results when gold isused as the sensor surface. Such an improvement in the sensitivity limitshould allow SPR biosensors to become a possible replacement forconventional biosensing techniques based on fluorescence.

In conclusion, the present invention has many advantages. First, itoffers higher sensitivity by increasing the phase shift. Second, thestability of phase measurement is enhanced because of differentialmeasurement by using a Wollaston prism. Third, the invention offers 2-Dmapping surface reactions in real time, which may find wide applicationin chemical, biochemical and biological processes. Fourth, the system issimple and can be manufactured at low cost.

While we have hereinbefore described embodiments of this invention, itis understood that our basic constructions can be altered to provideother embodiments which utilize the processes and compositions of thisinvention. Consequently, it will be appreciated that the scope of thisinvention is to be defined by the claims appended hereto rather than bythe specific embodiments which have been presented hereinbefore by wayof examples.

1. A method of detecting biological biochemical, or chemicalcharacteristics of a species comprising: providing a device comprisingan SPR sensor having a sensing surface; allowing a fluid including thespecies through a channel attached to the sensing surface; subjecting alight to passing through the SPR sensor so that the light is reflectedat least twice by the sensing surface; obtaining a two-dimensionalinterference fringe pattern of the light back from the SPR sensor;measuring a differential phase between a p-polarization and ans-polarization of the light through analyzing the two-dimensionalinterference fringe pattern; and determining the biological,biochemical, or chemical characteristics of the species in accordancewith the differential phase.
 2. The method of claim 1, wherein themeasuring of a differential phase comprises: averaging an interferencefringe pattern of the light to produce a profile of an intensityvariation for each of a p-polarization and an s-polarizationinterference fringe patterns; extracting an intensity distribution ofeach of the p-polarization interference fringe pattern and thes-polarization interference fringe pattern respectively from the profileof the intensity variation to obtain a curve of the intensitydistribution of each of the p-polarization and s-polarizationinterference fringe patterns; differentiating the curves respectively tofind a peak of each of the curves; and obtaining the differential phasethrough a location of the peak of each of the curves.
 3. The method ofclaim 2, wherein the averaging of an interference fringe patterncomprises: reading a value of a pixel over a pre-defined rectangularregion within the pattern; separating the rectangular region intoone-pixel wide vertical traces, each containing a different intensityvalue; finding a mean horizontal pixel value of each of all the verticaltraces; and producing the profile of the intensity variation from themean horizontal pixel values.
 4. The method of claim 2, wherein theobtaining the differential phase comprises: comparing a location of thepeak of each of the two curves, if the location is an integer pixelvalue; or locating the peak as a fraction of pixel by means of linearextrapolation using the intensity value of two adjacent pixels, if alocation of the peak is not an integer pixel value, and comparing thelocation of the peak of each of the two curves.